The present invention is directed to improved methods and immunoassays for detecting two or more ligands in a liquid sample suspected of containing such ligands, and more particularly, improved methods and immunoassays for rapidly and qualitatively detecting the presence or absence of one or more ligands via a single, rapid, competitive ligand-receptor reaction.
Immunoassays, also referred to as ligand-receptor assays, are well-known in the art. Since their introduction in 1971, immunoassays have been widely utilized in the medical field as a diagnostic tool to detect minute amounts of hormones, drugs, antibodies and other substances suspected of being present in a given fluid sample. Immunoassays have found further application in determining the presence and concentration of ligands in food products and environmental samples.
Such assays rely on the binding of ligands by receptors to determine the concentration of such ligands in a given sample and are typically characterized as either competitive or non-competitive. Non-competitive assays generally utilize receptors in substantial excess over the concentration of ligands to be determined in the assay. Typical of such non-competitive immunoassays include sandwich assays, which detect the presence of a ligand by binding two receptors thereto. In such arrangement, the first antibody is bound to a solid phase such that when the ligand is present, such ligand becomes affixed thereto. A second receptor having a label covalently attached thereto, which may comprise a radioactive, fluorescent, enzymatic, dye or other detectable moiety (collectively referred to as tracers), is introduced to the assay which consequently binds to the bound ligand, to the extent the ligand is present, and thereafter produces a signal consistent with the presence of such ligand. If the sample does not contain the molecules of interest, the labeled antibody is carried past the immobilized antibody without reacting which, as a consequence, will not cause a change in the membrane. Such non-competitive immunoassays are primarily useful for the detection of large molecules such as proteins, large hormones or molecules which have multiple binding sites, such as human chorionic gonadotropin (HCG) and typically will not work with small molecules that have only one binding site.
Competitive assays, in contrast, generally involve competition between a ligand present in a given sample, and a ligand analog having a tracer/label covalently linked thereto to permit detection for a limited number of binding sites provided by the ligand receptor, which typically comprises an antibody bound to a solid phase. Such assays are particularly suited to detect smaller molecules, such as drugs and drug metabolites. In this context, drug analogs are utilized that have been covalently bound to a protein which is then immobilized on a membrane. Antibody specific to the drug is then labeled and immobilized on a porous pad. When a sample is added which is suspected of containing a given analyte, such sample dissolves the labeled antibody and carries it into contact with the immobilized drug-protein region. If there is little or not drug in the sample, a large amount of the labeled antibody is bound to the immobilized drug-protein region which, consequently, produces a detectable signal. If the sample contains a high amount of drug, little or no labeled antibody is bound to the immobilized drug-protein region and thus in turn gives little or no signal.
Early immunoassays required tedious manual steps and long incubation times, typically lasting for several hours. Recent advancements in immunoassays, however, have overcome such deficiencies and can now allow an immunoassay to be formed in less than ten minutes. Such immunoassays may further typically be performed with only one step, which typically comprises mixing all the reactants of a competitive ligand-receptor assay, namely, a fluid sample suspected of containing the ligand, a labeled ligand analog, and a receptor (antibody) bound to a solid phase, with the quantity of ligand being determined by its effect on the extent of binding between the ligand receptor and the labeled ligand analog.
Today, rapid immunoassays generally consist of an adhesive-covered plastic backing onto which several porous pads and a piece of protein-binding membrane are attached. The membrane typically contains a section that has been impregnated with a binding partner (i.e., a receptor or ligand analog). A second pad is typically provided which contains a labeled target molecule or labeled antibody (i.e., tracer) that is placed in direct contact with the protein-binding membrane. When a sample suspected of containing a target ligand is contacted with the immunoassay, such sample dissolves the labeled element or tracer and the capillary action of the protein-binding membrane subsequently draws the sample with tracer dissolved therein into contact with the impregnated binding partner. When this reaction occurs, there is a change in the appearance of the binding membrane, with the difference providing a qualitative and quantitative indication of the presence or absence of the ligand suspected of being present in such sample.
While today's rapid competitive immunoassays are considered generally effective, such assay methods continue to suffer from significant drawbacks. In particular, the reaction between the labeled antibody and the drug in a sample, to the extent present, is known to begin prior to when the labeled antibody reaches the immobilized drug-protein region. Moreover, because the reaction time for such assays is dependent upon variables, such as sample viscosity and membrane porosity, such variability in time allows more or less reaction between the labeled antibody and sample which decreases the sensitivity of the assay and gives rise to inconsistent results.
Accordingly, there is a substantial need in the art for a rapid immunoassay that, in addition to providing a rapid qualitative indication as to whether or not a specific ligand is present within a given sample, has greater sensitivity and reproduceability than prior art assays and methods. Specifically, there is a need for rapid immunoassays and assay methods that can be performed in a single step that further provides qualitative results by utilizing a single competitive assay between a ligand and ligand analog with binding sites to a bound receptor. In addition, there is a need in the art for an immunoassay and assay method that are inexpensive, relatively easy to manufacture, and capable of being utilized for a wide variety of applications. There is still further a need for a rapid, single-step competitive immunoassay that can identify the presence of two or more suspect ligands in a given sample.